Enzyme Linked Immunosorbent Assay (ELISA)
Elisa stands for enzyme linked immunosorbent assay.
ELISAs follow the basic principle of an antigen binding to its specific antibody. This allows for the detection of analytes within samples, even if present in small amounts. ELISAs can be used for the detection of hormones, peptides, proteins and small molecules and can be used for medical diagnostics and analyte measurement in industry and in research. Various types of ELISAs have been developed with some alterations to their basic steps. However, they all still use the basic principle that an antibody will bind to a specific epitope on an antigen. The use of an enzyme and substrate, often TMB and HRP, are used to produce a colour change can then be used to determine the concentration of analyte present within a sample. Measuring the OD absorbance values and producing a standard curve from known antigen concentrations allows for the concentration of the analyte to be calculated.
The four major types of ELISA are indirect, direct, sandwich and competitive.
These are considered the simplest form of ELISA. They are referred to as direct as the use of only one antibody is required for the detection process. The sample is coated onto the plate and the plate is subsequently blocked. The primary detection antibody is directly labelled with a conjugated enzyme which produces a colour change when substrate is added. No secondary antibody is required. When testing for the analyte of interest within a sample a blocking agent such as BSA must also be used to block off any other potential binding sites. Direct ELISAs are recognised to reduce cross reactivity between other antibodies as only one is used. However, as the primary antibodies used within these assays need to be labelled with enzyme this can increase the cost of running multiple assays and the time required to produce them. With each new assay, a new detection antibody would need to be labelled. Direct ELISAs are recognised to have a lower sensitivity as the signal produced from the assay is less amplified when compared to indirect ELISAs; however, they can be performed much faster as only one step is required for detection.
Indirect ELISAs require the use of two antibodies for detection. This involves a two step process, increasing the time involved in carrying out the assay. The antigen of interest is first coated on to the plate , the primary antibody will then be introduced to the wells. The primary antibody will specifically bind to the antigen of interest if present in the wells. Following this the secondary antibody will be introduced, this secondary antibody will have been previously labelled with an enzyme for detection. The secondary antibody will bind to the primary antibody which will in turn be bound to the antigen. The concentration of antigen can then be determined depending on a colour change observed. The more antibody bound to antigen the greater the colour change as less will be removed following the wash cycles. The benefits to this process can include a higher sensitivity as more than one labelled antibody can bind to the primary antibody. Flexibility may also be increased as more than one secondary detection antibody can be used with a single primary detection antibody. There may also be a reduction in cost to perform this assay as only one type of antibody will need to be labelled.
Most often used to determine small molecules such as lipids, hormones and small peptides. Larger molecules may also be detected however larger concentrations would be required.
Competitive Elisa's follow the principle that the sample antigen of interest and an enzyme conjugated version of the same antigen will compete with each other for limited numbers of specific antibody binding sites. Another method can be the antibody competes for target sites on the bound antigen pre coated to the plate. Labelled antibody will compete for binding sites with the antibody within the sample.
When detecting the concentration of antibody in a sample the lower the signal produced from the assay the higher the level of specific antibody within the sample. The known labelled antibody will compete for binding with the antigen pre coated to the wells, if more of the antibody in the sample is able to bind this will result in more of the labelled antibody being removed following the wash cycles. For labelled antigen this follows the same principle.
Sandwich ELISAs tend to be the most readily recognized, and as the name suggests the antigen of interest will be sandwiched between two antibodies, these assay may either be direct or indirect.
As the antigen is sandwiched between two antibodies the antigen of interest is required to be fairly large in size, usually larger than 20aa in length. This ensures that the antibodies used are able to bind to different epitopes of the antigen and will not disrupt the binding of each of the antibodies used. The capture antibody is the antibody used to absorb the target antigen onto the wells of the assay. Once the sample has been added the detection antibody may then be introduced to the wells, binding to the target antigen if present in the wells. When carrying out a sandwich Elisa it is important that the antibodies used are matched pairs. Matched pairs refers to antibodies being specifically tested together to ensure that they bind to different epitopes of an antigen. This prevents the chance of the antibodies binding to the same site or recognising each other. The detection antibody within the sandwich Elisa may be enzyme conjugated or a secondary antibody may be added that is enzyme conjugated. Enzymes used within the detection process more often include HRP (horse radish peroxidase) or AP (alkaline phosphatase). The final step in the process is the addition of the substrate. The substrate added will react with the enzyme conjugated antibody to produce a reaction that often involves a colour change, the release of light or fluorescence. The signal strength can be detected using specific absorbency readers. A stop solution must be added during this procedure to halt the reaction over a specified period of time otherwise the signal will become saturated/ degrade.
Sandwich Elisa's are beneficial when it is suspected that the samples may have low levels of antigen present. This is due to the capture antibody only binding to the antigen of interest. The other proteins within the sample that have not bound to the capture antibody will be subsequently washed away.
ELISAs may be run as either qualitative or quantitative. Qualitative results provide either a positive or negative result for the sample. The cut off between positive and negative will be determined by the end user. In quantitative elisas however the amount of coloured product observed will be directly proportional to the amount of enzyme linked antibody that has bound to the primary antibody in turn bound to the antigen. The concentration of antigen/antibody within a sample can be determined from standards used which have known amounts of antibody or antigen. A standard curve is produced from serial dilutions of the standard and the absorbency values they produce. Using the graph the concentration of the antibody/antigens can be determined when taking into account their absorbency values.
Blocking buffers are used to prevent non specific binding within the assay. Proteins and other bio molecules may bind to unoccupied spaces on the surface of the wells which can be detrimental to the specificity and sensitivity of the assays results as less of the antigen of interest will be able to bind. Non specific binding can be diminished through the use of a blocking buffer. The two major classes of blocking reagents are proteins and detergents. the key aspects to consider when chosing a blocking reagent is making sure it exhibits no cross reactivity with other components of the kit, helps to stabilise biomolecules minimising the effects of denaturation that can be caused from assay transitions, exhibit low enzyme activity to prevent interference with the detection method.
This is one of the most commonly used blocking agents. Tween 20 is a non ionic detergent that is able to block areas on the surface that may be exposed. The benefit to these are they are inexpensive, extremely stable and can be used in washing solutions. The disadvantages are they are not permanent, they may disrupt non covalent bonds, they are only able to block hydrophobic interactions.
Is a type of protein blocker, unlike detergent blockers protein blockers are permanent and only need to be introduced into the wells once when the capture antibody has been added. Protein blockers block the unoccupied spaces on the surface and also stabilise the biomolecules bound to the surface helping to reduce denaturation. Other forms of protein blockers include non ft dry mil, whole natural serum and fish gelatin.
The type of blocker chosen is also dependent on the surface you are blocking.